Production of transgenic piglets using ICSI–sperm-mediated gene transfer in combination with recombinase RecA

Abstract

Sperm-mediated gene transfer (SMGT) is a method for the production of transgenic animals based on the intrinsic ability of sperm cells to bind and internalize exogenous DNA molecules and to transfer them into the oocyte at fertilization. Recombinase-A (RecA) proteincoated exogenous DNA has been used previously in pronuclear injection systems increasing integration into goat and pig genomes. However, there are no data regarding transgene expression after ICSI. Here, we set out to investigate whether the expression of transgenic DNA in porcine embryos is improved by recombinase-mediated DNA transfer and if it is possible to generate transgenic animals using this methodology. Different factors which could affect the performance of this transgenic methodology were analyzed by studying 1) the effect of the presence of exogenous DNA and RecA protein on boar sperm functionality; 2) the effect of recombinase RecA on in vitro enhanced green fluorescent protein (EGFP)-expressing embryos produced by ICSI or IVF; and 3) the efficiency of generation of transgenic piglets by RecA-mediated ICSI. Our results suggested that 1) the presence of exogenous DNA and RecA-DNA complexes at 5 mg/ml did not affect sperm functionality in terms of motility, viability, membrane lipid disorder, or reactive oxygen species generation; 2) EGFP-expressing embryos were obtained with a high efficiency using the SMGT-ICSI technique in combination with recombinase; however, the use of IVF system did not result in any fluorescent embryos; and 3) transgenic piglets were produced by this methodology. To our knowledge, this is the first time that transgenic pigs have been produced by ICSI-SGMT and a recombinase.

Hyperactive self-inactivating piggyBac for transposase-enhanced pronuclear microinjection transgenesis

“…Only a few species are amenable to the micromanipulations necessary for efficient ICSI-Tr. For example, ICSI-Tr is inefficient in pigs, and therefore appears unsuitable for routine transgenic production (34)(35)(36). Lentiviral-mediated transgenesis, on the other hand, is very efficient in delivering the transgene to the host genome but results in multicopy insertions; as a consequence, only a few micromanipulated embryos reach term, probably due to lethal mutagenesis.…”

Genetic Modification of Domestic Animals for Agriculture and Biomedical Applications

“…N um e r ou s met h o d s h a ve bee n successfully used to introduce genetic modifications and produce transgenic animals, including pronuclear microinjection of foreign DNA into zygotes (Hammer et al, 1985;Pursel and Rexroad, 1993), viral-mediated gene transfer (Chan et al, 1998;Cabot et al, 2001;Whitelaw et al, 2008), sperm-mediated gene transfer (Castro et al, 1991;Chang et al, 2002;Lavitrano et al, 2002 and and intracytoplasmic injection (ICSI) of a sperm head carrying foreign DNA (Perry et al, 1999;Osada et al, 2005;Moisvadi et al, 2009;García-Vázquez et al, 2010). Despite the proven successful application of these techniques, some problems, such as inefficiency and mosaicism (transgene not going into the germline) (Table 1) remain to be solved and limit the practical application of these methods.…”

Intracytoplasmic sperm injection in domestic and wild mammals

“…Moreover, after the generation of the first transgenic piglet by sperm injection, ICSI gained importance as a new tool for inducing genetic modifications in farm animals (Kurome et al 2006, Garcia-Vazquez et al 2010. However, the advent of CRISPR-Cas9 system for genetic engineering lead to the replacement of ICSI by regular or IVF zygotes for the generation of genetically modified animals (Hai et al 2014, Whitworth et al 2014, Proudfoot et al 2015, Wang et al 2015, Bevacqua et al 2016.…”